As used herein, septic shock refers to a systemic immune system dysfunction in response to an overwhelming infection leading to hypotension and organ failure (1). Over 750,000 cases of severe sepsis (the precursor to shock) occur each year in the United States with an overall mortality rate of 28%, making the number of deaths similar to that from coronary artery disease (2). Because early intervention with supportive therapies makes a difference in outcome (3), means to prospectively stratify patients on the basis of risk has become one of the central objectives in the sepsis field (1, 4). The failure of numerous clinical trials of immunomodulatory therapeutics over the past few decades, some of which actually show higher mortality, highlights the long felt need for improved prognostic indicators. These trials demonstrate that there is significant patient-to-patient diversity of immune responses during severe sepsis.
A novel signaling pathway pertinent to sepsis pathophysiology has recently been identified with global control over monocyte and macrophage inflammatory mediator production and microbial killing (5, 6). Specifically, extracellular adenine nucleotides, such as ATP, are released systemically by the adrenal gland, as well as locally by platelet degranulation and/or by cell death during the inflammatory response in sepsis. These hormones modulate monocyte and macrophage immune responses via interaction with the nucleotide receptor P2X7 (5).
The P2X7 receptor controls the production of inflammatory mediators during sepsis, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, nitric oxide (NO), tissue factor, and prostaglandins (7-12). P2X7-knockout mice exhibit greatly attenuated production of IL-1beta and IL-6 in response to endotoxin (lipopolysaccharide, LPS) (11), a common pathogenic agent in severe sepsis. Additionally, P2X7 stimulation promotes membrane fusion events such as phagolysosomal maturation necessary for microbial killing, microvesicle generation required for IL-1beta processing, and giant cell formation needed to make granulomas (13-15). Finally, co-administration of the ATP analogue, 2-methylthio-ATP, protects mice from endotoxic death in an animal model of severe sepsis with concomitant reductions in LPS-induced serum levels of TNF-alpha and IL-1 (7). Thus, extracellular adenine nucleotides and the nucleotide receptor P2X7 have a profound influence on monocyte and macrophage immune responses relevant to sepsis pathophysiology.
The family of P2 receptors binds extracellular nucleotides with two or more phosphates and has been divided into the P2X and P2Y subfamilies according to whether the individual member acts as an ion channel or a G-protein coupled receptor, respectively (6). P2X7 belongs to the P2X family due to its structural similarity with the six other members, each having two predicted membrane spanning domains (6, 16). Whereas ligand-gated, nonselective cation channel activity is a common feature of the P2X family, reversible permeability to larger molecules (<900 Da) is a feature more characteristic of P2X7 under biological conditions (6).
The gene for human P2X7 contains two previously-described single nucleotide polymorphisms (SNPs) associated with functionally significant amino acid substitutions. Gu et al. have shown that the human P2X7 gene contains a nucleotide polymorphism (SNP, A1513C) conferring an amino acid substitution that disrupts the pore activity of this receptor. In addition, Wiley et al. report that a T1729A mutation is associated with reduced pore activity due to a trafficking defect (Wiley et al. J. Biol. Chem. 278:17108-17113 (2003)).
Because the P2X7 pore activity has been linked to monocyte and macrophage inflammatory mediator production (particularly IL-beta (14)), and because inflammatory mediator production is a major determinant in deciding on courses of immunosuppressive and anti-inflammatory therapies, it is particularly desirable to obtain a rapid and convenient clinical assay for determining P2X7 pore activity. A rapid assay of P2X7 pore activity is required to make reliable prognoses and refined therapeutic interventions.
Unfortunately, presently-known P2X7 pore assays do not provide rapid and robust procedures for use outside of the laboratory setting, most notably in the clinical setting. For example, Gu et al., J. Biol. Chem. 276, 11135-11142, describe the A1513C polymorphism and provide a P2X7 pore assay based on ethidium bromide uptake in ATP-induced monocytes. A similar assay was utilized by Wiley et al. in analyzing the T1729A polymorphism. However, this assay requires extensive isolation and purification of these cells apart from other cell types before dye influx can be measured by time-resolved flow cytometry. In particular, this method requires the use of a ficoll hypaque density gradient to obtain the necessary monocytes. The preparatory step is therefore time-consuming and, due to the technical aspects related to density gradient separation, not practical in the clinical setting where complex bench and cold room facilities are not available. The time necessary to carry out this technique is estimated to be at least one full workday for one skilled in the field with multiple sample processing not easily amenable to automation. Moreover, the volume of blood needed for the previous P2X7 pore assay (i.e., several hundred cc's) precludes testing in pediatric and frail subjects.
As well, Patent Application US 2002/0182646 A1, published Dec. 5, 2002 to Ke et al. describes a method for measuring P2X7 receptor-mediated macromolecule uptake in macrophages. Like the method of Gu et al. discussed above, this approach also relies on complex preparatory steps to provide isolated and purified macrophages before pore activities may be reliably measured. Specifically, Ke et al. teach that macrophages are harvested from the peritoneal cavity of animals (e.g., mice) by medium injection into the cavity, followed by collection of the lavage fluid. Quite obviously, this approach does not provide a practical clinical procedure for rapid measurement of P2X7 pore activity in humans.
Based upon the above-described needs and others, it is therefore desirable to obtain a rapid P2X7 pore assay suitable for, but not limited to, use in the clinical setting. This assay would dispense with the time-consuming and technical complexities of previous methods. Preferably, the assay could be carried out directly on clinical specimens, such as, whole blood samples. Furthermore, the assay would provide improved sensitivity, reliability and robustness while, at the same time, being amenable to automation.